Week Two: The Waiting Game

   This last week consisted of lots more PCRs and cloning samples for sequencing, and while both of these are fantastic tools that work efficiently, they can be long and somewhat tedious, especially when dealing with 50 samples.

During Week One I ran several samples using my ORF primers - essentially, we wanted to see if these primers could amplify any Unique Sequence Elements (USE) that could be on particular interest in Peter's lab.  Pete and I selected 4 samples to clone (times ten, as there were several bands), meaning I had at least 40 samples to clone and prep for sequencing.  On top of that, three of my Ilwaco Harbor samples for the Euduboscquella PCR (Dub) worked, so I cloned those as well to make sure the right sequence was being amplified.  50 samples may not sound like a lot, but that took up the majority of my time last week.

Along with cloning and prepping for sequencing, I also ran some more Dub PCRs for October samples (the end of red water, we wanted to see if this dinoflagellate only appears with RW) and a Willapa Port sample (also red water, but further removed from the other three sample locations).  As I suspected, the decline of red water also meant a decline in signal from our dinoflagellate, meaning I have to run more PCRs on non-red water samples to see if our suspicions are true.  I also got a signal from the red water Willapa sample, indicating we are going in the right direction.

This week, I will be doing more PCRs and analyzing the sequences I got back, as well as doing something called FISH microscopy.  FISH, or Fluorescence in situ Hybridization is a way to look at preserved cells under a microscope.  We use two stains caled DAPI (which stains DNA and RNA) and proflavine (which stains cytoplasm and fluoresces different colors depending on if the cell is an autotroph or heterotroph).  I'm really excited to do this as I haven't had much experience with microscopy before.

Until next time, Deirdre